Assays and enhancers of the human delta enac sodium channel

ABSTRACT

This invention relates to electrophysiological assays that measure sodium conductance activity of a delta human epithelial sodium channel (ENaC) in the presence and absence of delta hENaC enhancers. Also, the invention generally relates to assays for identifying compounds that enhance the activity of delta hENaC, especially in an oocyte expression system. These compounds have potential application in modulating (enhancing) salty taste perception.

RELATED APPLICATIONS

This application claims benefit of priority to and incorporates byreference in its entirety U.S. provisional application No. 60/764,353filed on Feb. 2, 2006 by Bryan Moyer et al.

FIELD OF THE INVENTION

This invention relates to electrophysiological assays that identifycompounds that modulate a human sodium epithelial channel comprised ofdelta, beta and gamma subunits in the presence and absence of ENaCenhancers and the use thereof to modulate human salty taste perception.The invention more specifically relates to the identification ofcompounds that enhance a human ENaC comprised of delta, beta and gammasubunits expressed in an oocyte expression system. Further the inventionrelates to assays for identifying compounds that modulate a human ENaCcomprised of delta, beta and gamma subunits and the use thereof tomodulate human salty taste perception.

As described herein electrophysiological assays conducted using humanENaC comprised of either alpha, beta and gamma subunits or delta, betaand gamma subunits have shown that amiloride blocks delta beta gammaENaC, ˜25-fold less efficiently than alpha beta gamma ENaC. Unlike othermammals, amiloride only slightly reduces the intensity of sodiumchloride taste, i.e., by about 15-20% when used at concentrations thatspecifically modulate ENaC function. Experiments conducted by theinventors have shown that amiloride did not elicit a significant effecton perceived salt intensity when tested at levels ˜300-fold above IC50values for alpha beta gamma ENaC in oocytes (equivalent to only ˜10-foldover IC50 values for delta beta gamma ENaC in oocytes).

Based thereon, assays have been developed which are disclosed herein toidentify compounds that modulate the delta beta gamma human ENaC sinceit is anticipated that these compounds will potentially modulate humansalty taste perception.

BACKGROUND OF THE INVENTION

Epithelial sodium channels (ENaC) are members of the ENaC/degenerinfamily of ion channels that includes acid-sensing ion channels (ASIC) inmammals, mechanosensitive degenerin channels in worms, and FMRF-amidepeptide-gated channels in mollusks (Kellenger, S. and Schild, L. (2002)Physiol. Rev. 82:735-767). ENaC mediates apical membrane Na⁺ transportacross high resistance epithelia in numerous tissues including kidney,colon, and lung.

ENaC is known to be a heterotrimeric channel comprised of α, β, and γsubunits. This heterotrimeric channel has been hypothesized to beinvolved in human salty taste perception. Additionally, this channel isinvolved in the maintenance of extracellular volume and blood pressure,absorption of fluid from the lungs during late stages of gestation, andtransduction of salt taste. (See e.g., Rossier, B. C. et al., Annu. Rev.Physiol. 62:877-897 (2002); Alvaraz et al. Annu. Rev. Physiol.62:573-594 (2000); and Bigiani et al., Prog. Biophys. Mol. Biol.83:193-225 (2003)).

For example, it is known that mutations in the human ENaC (hENaC),particularly gain of function mutations result in hypertension due toincreased renal Na⁺ reabsorption in Liddle's syndrome (Schild et al.,Proc. Natl. Acad. Sci., USA 92:5699-5703 (1995); Shimkets et al., Cell79:407-414 (1994); and Snyder et al., Cell 83:969-98 (1995)). Bycontrast, it is known that hENaC loss of function mutations result insalt-wasting due to decreased renal Na⁺ reabsorption inpseudohypoaldosteronism type I (PHA1). (See Grunder et al., EMBO. J.16:899-907 (1997); and Chang et al., Nat. Genet. 12:248-253 (1996)). Theclinical symptoms of salt-wasting include by way of examplehyponatremia, hyperkalemia, dehydration, elevated serum aldosterone, andmineralocorticoid unresponsiveness.

BRIEF DESCRIPTION AND OBJECTS OF THE INVENTION

In this invention disclosure we describe screening assays to identifyhuman delta epithelial sodium channel (ENaC) enhancers.

It is a specific object of the invention to provide electrophysiologicalassays that measure sodium conductance of delta beta gamma human ENaCchannels in the presence and absence of delta ENaC enhancers.

It is another specific object of the invention to provide enhancers ofthe delta beta gamma human ENaC channels in an oocyte expression system.

It is another object of the invention to identify delta ENaC specificenhancers that modulate, preferably enhance human salty tasteperception.

More preferably it is an object of the invention to provide patchclamping or two electrode voltage clamping assays using oocytes thatexpress a human delta beta gamma ENaC channel for identifying compoundsthat modulate the activity of this channel.

As described infra, experiments performed by the inventors have notdemonstrated a significant effect of amiloride on perceived saltintensity when tested at levels ˜300-fold above the IC50 value for alphabeta gamma ENaC in oocytes (equivalent to ˜10-fold over the IC50 valuefor delta beta gamma ENaC in oocytes). Since delta ENaC is ˜25-fold lesssensitive to amiloride than alpha ENaC, and human salt taste is poorlyinhibited by amiloride, it is believed that human salt taste may bemediated, in part, by a delta ENaC-based sodium channel. Thus, theinvention provides assays for identifying modulators of the human deltachannel which may be comprised of a delta beta gamma heterotrimer, adelta only monomer, or any combination of delta with beta and gamma orother protein subunits. The compounds identified and their derivativespotentially can be used as modulators of human salty taste in foods,beverages and medicinals for human consumption.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B compares human alpha beta gamma and human delta betagamma ENaC channel function in oocytes in the presence of amiloride.

FIGS. 2A and 2B contains representative traces of oocytes expressingwild-type alpha beta gamma hENaC (top traces) and delta beta gamma hENaC(bottom traces) stimulated with amiloride and a proprietary compoundidentified by the present Assignee Senomyx Inc. as an ENaC enhancer.

FIG. 3 shows taste-cell specific expression of delta ENaC in monkey CVtaste tissue by PCR screening.

FIG. 4 contains the results of in situ hybridization experiments showingdelta ENaC mRNA expression in a subset of monkey CV taste cells.

SUMMARY OF INVENTION

The subject invention relates to screening assays for identifying humandelta epithelial sodium channel (ENaC) enhancers.

As described supra, an inhibitor of ENaC sodium channel function,amiloride, attenuates gustatory responses to sodium chloride in numerousnon-mammalian as well as mammalian species, including rodents but nothumans. In humans, amiloride has been reported to reduce the intensityof sodium chloride by only 15-20% when used at concentrations thatspecifically inhibit ENaC function. Experiments performed at Senomyx didnot demonstrate a significant effect of amiloride on perceived saltintensity when tested at levels ˜300-fold above IC50 values for alphabeta gamma ENaC in oocytes (equivalent to ˜10-fold over IC50 values fordelta beta gamma ENaC in oocytes). Since delta ENaC is ˜25-fold lesssensitive to amiloride than alpha ENaC, and human salt taste is poorlyinhibited by amiloride, human salt taste may be mediated, in part, by adelta ENaC-based sodium channel. Thus, experiments described infra wereused as the basis for the development of novel delta ENaC-based assaysto identify delta ENaC enhancers.

Molecular Biology—

α, β, and γ hENaC were cloned from kidney cDNA (Origene, Rockville, Md.)into pcDNA3 (Invitrogen, Carlsbad, Calif.) as described previously(Kellenberger, S and Schild, L. Physiol. Rev. 82:735-767(2002). 5 hENaCwas cloned from testis cDNA (BD Biosciences Clontech, Palo Alto,Calif.). α hENaC sequence was identical to published sequences from lungand kidney (Rossier et al., Annu. Rev. Physiol. 64:877-897 (2002);Alvarez de la Rosa et al., Annu. Rev. Physiol. 62:573-594 (200).(Genbank accession numbers X76180 and L29007). β hENaC sequence wasidentical to published sequence from lung Bigiani et al. Bigiani et al.,Prog. Biophys. Mol. Biol. 83:193-225 (2003). (Genbank accession numberX87159) with the exception of a glycine (nucleotide triplet GGC) toalanine (nucleotide triplet GCC) substitution in our clone at amino acid314. Inspection of the public single nucleotide polymorphism (SNP)database revealed that glycine 314 and alanine 314 are polymorphisms inβ hENaC. γ hENaC sequence was identical to published sequence fromplacenta (Schild et al., Proc. Natl. Acad. Sci., USA 92:5699-5703(1995)). (GenBank accession number BC059391). δ hENaC sequence wasidentical to published sequence from kidney (Shimkets et al., Cell 79:407-414 (1994)) with the exception of a tyrosine (nucleotide tripletTAC) to cysteine (nucleotide triplet TGC) substitution in our clone atamino acid 532. Inspection of the human genome revealed cysteine atamino acid 532, and the public SNP database lists cysteine 532 andtyrosine 532 as polymorphisms in δ hENaC.

In Vitro Transcription—

ENaC cRNA was generated from linearized plasmids using the mMESSAGEmMACHINE kit with T7 RNA polymerase according to the manufacturer'sinstructions (Ambion, Austin, Tex.). cRNA quality was checked bydenaturing agarose gel electrophoresis and spectrophometric absorbancereadings at 260 and 280 nm to ensure that full-length, non-degraded cRNAwas generated.

Frog Surgery and Oocyte Isolation—

Female Xenopus laevis South African clawed frogs greater than or equalto 9 cm in length were obtained from NASCO (Fort Atkinson, Wis.). Frogswere anesthetized in 0.15% ethyl-3-aminobenzoate methanesulfonate(Sigma, St. Louis, Mo.) in distilled water and placed on ice. Usingsterile surgical tools, sequential 1-2 cm incisions were made in theabdomen through both the outer skin layer and the inner peritoneal layerto revel the ovaries. Excised ovarian lobes (containing 1000-2000oocytes) were placed in OR-2 calcium-free media (82.5 mM NaCl, 2 mM KCl,1 mM MgCl₂, 5 mM HEPES pH 7.5 with NaOH) and sequentially digested with2 mg/ml collagenase type IA (Sigma), prepared immediately before use,for 45 min followed by 1 mg/ml collagenase type IA for 15 min on arocking platform at room temperature. After enzymatic digestion, atwhich point the majority of oocytes are released from the ovarian lobes,oocytes were thoroughly rinsed in OR-2 without collagenase andtransferred to a Petri dish containing Barth's saline (88 mM NaCl, 2 mMKCl, 0.82 mM MgSO₄, 0.33 mM Ca(NOs)₂, 0.41 mM CaCl₂, 2.4 mM NaHCO₃, and5 mM HEPES pH 7.5; Specialty Media, Phillipsburg, N.J.) supplementedwith 2.5 mM sodium pyruvate. Mature stage V or VI oocytes (˜1 mmdiameter) were selected for microinjection. Frogs were sutured using aC6 needle with a 3-0 black braid suture (Harvard Apparatus, Holliston,Mass.) and reused for subsequent oocyte isolation; following a 2-3 monthrecovery period.

Microinjection—

Microinjection needles were pulled on a Model P-97 Flaming/BrownMicropipette Puller (Sutter Instrument Co., Novato, Calif.) usingborosilicate glass capillaries (World Precision Instruments, Sarasota,Fla.), back-filled with mineral oil (Sigma), and then front-filled withENaC cRNA using a Nanoliter 2000 injector with a Micro4 MicroSyringePump Controller (World Precision Instruments). Oocytes weremicroinjected in the animal pole with 10-15 nl containing 1 ng of eachENaC subunit cRNA. Following microinjection, oocytes were incubated inBarth's solution supplemented with 2.5 mM sodium pyruvate at 18° C.overnight.

Two Electrode Voltage Clamping—

Unless noted otherwise, ENaC function was measured using thetwo-electrode voltage clamp technique on an OpusXpress 6000A paralleloocyte voltage clamp system twenty-four hours post-microinjection (AxonInstruments, Union City, Calif.). The OpusXpress system is an integratedworkstation that allows electrophysiological recordings to be made fromup to 8 oocytes simultaneously. This system has previously been used toexamine the function of ion channels including nicotinic acetylcholineand serotonin 5HT3 receptors Snyder et al., Cell 83:969-978 (1995);Grunder et al., Embo J. 16; 899-907 (1997); Chang et al., Nat. Genet.12:248-253 (1996); Zennaro et al., Trends Endrocrinol. Metab. 15:264-270(2004); and Bonny et al., Pediatr. Nephrol. 17:804-808 (2002). Oocyteimpalement is automated and compound delivery is performed by acomputer-controlled fluid handler; compounds are removed from 96-wellplates using disposable pipette tips and applied to individual oocytes.Oocytes were placed in the OpusXpress system and perfused with ND-96solution (96 mM NaCl, 2.5 mM KCl, 1 mM CaCl₂, 1 mM MgCl₂, and 5 mM HEPESpH 7.5 with NaOH). Oocytes were then impaled with voltage-sensing andcurrent-sensing electrodes back-filled with 3M KCl. Electrodes exhibitedresistances between 2-10 Mohm for voltage-sensing electrodes and between0.5-2 Mohm for current-sensing electrodes. Following impalement, oocyteswere voltage clamped to −60 mV and experimental recordings wereinitiated. Data were acquired at 50 Hz and low-pass filtered at 5 Hz.

Compounds—

The proprietary enhancer compound 6363969 was diluted to appropriateconcentrations in ND-96 from 100 mM stock solutions in DMSO. The finalconcentration of DMSO in experiments was <0.1%; this level of vehiclehad no effect on ENaC function in oocytes.

Statistics and Measurements—

Data represent the mean+/−SEM. Unless otherwise noted, experiments wereperformed on two to four batches of independently injected oocytesharvested from different frogs. Statistical significance betweendifferent groups was determined using an unpaired, two-tailed Student'st-test. Dose-response curves were plotted and both EC₅₀ values and Hillcoefficients were determined using GraphPad Prism v3.02 (GraphPadSoftware, San Diego, Calif.). Values for percent hENaC activation werecalculated by dividing the magnitude of the inward current induced byENaC enhancer compounds by the magnitude of the inward curren blocked byamiloride in the same oocyte and multiplying the ratio by 100%.

Results—

Similar to a hENaC, 8 hENaC can form functional amiloride-sensitivechannels when expressed alone or in combination with βγ hENaC; howeverδβγ hENaC is more than an order of magnitude less sensitive to amiloridecompared to αβγ hENaC Shimkets et al., Cell 79:407-414 (1994); and Bonnyet al., J. Clin. Invest. 104:967-974 (1999). a hENaC (human ENaC) and δhENaC are 58% identical at the DNA level and 35% identical at theprotein level. Expression of δβγ hENaC generated amiloride-sensitivecurrents; the IC₅₀ for amiloride inhibition of δβγ hENaC was 2.7+/−0.3uM (n=10), similar to previous reports Shimkets et al., (Id.); Bonny etal. (Id.), and much larger than the IC₅₀ for amiloride inhibition of αβγhENaC (110+/−11 nM; n=16) (FIG. 1). Activation of δβγ hENaC by 6363969was similar to αβγ hENaC (FIG. 1); the EC₅₀ for 6363969 activation ofδβγ hENaC was 1.2+/−0.2 uM (n=11) compared to the EC₅₀ for 6363969activation of αβγ hENaC of 1.2+/−0.1 uM (n=46). Representative tracesillustrating the effect of 6363969 on alpha and delta ENaC are shown inFIG. 2. Compounds from different chemical classes were tested on alphaand delta-based hENaC channels; all chemical classes activated alpha anddelta ENaC with similar efficacy and potency (Table 1).

Based on the foregoing, the present invention uses cell-based assays toidentify delta human ENaC modulators (enhancers). These compounds havepotential application in modulating human salty taste perception.Compounds identified in the subject electrophysiological assays andtheir biologically acceptable derivatives are to be tested in humantaste tests using human volunteers to confirm their effect on humansalty taste perception.

As discussed further infra, these cell-based assays preferably use highthroughput screening platforms to identify compounds that modulate(enhance) ENaC activity using cells that express human delta beta gammaENaCs. The sequences of these respective human delta, beta and gammasubunits are provided infra. Additionally, these sequences may bemodified to introduce silent mutations or mutations having a functionaleffect such as defined mutations that affect sodium ion influx. As notedabove, the assays will preferably comprise electrophysiological assayseffected in amphibian oocytes or assays using mammalian cells thatexpress a human delta beta gamma ENaC using fluorescent ion sensitivedyes or membrane potential dyes, e.g., sodium-sensitive dyes.Preferably, compounds that modulate ENaC are identified by screeningusing electrophysiological assays effected with oocytes that express ahuman delta beta gamma ENaC (e.g., patch clamping or two electrodevoltage clamping).

Still alternatively, compounds that modulate ENaC may be detected by ionflux assays, e.g., radiolabeled-ion flux assays or atomic absorptionspectroscopic coupled ion flux assays. As disclosed supra, these ENaCenhancers have potential application in modulating human salty tasteperception or for modulating other biological processes involvingaberrant or normal ENaC function.

The subject cell-based assays use mutant ENaC nucleic acid sequenceswhich are expressed in desired cells, preferably oocytes or human cellssuch as HEK-293 cells, or other human or mammalian cells conventionallyused in screens for identifying ion channel modulatory compounds. Thesecells may further be engineered to express other sequences, e.g., tasteGPCRs, i.e., T1Rs or T2Rs such as are described in other patentapplications by the present Assignee Senomyx. The oocyte system isadvantageous as it allows for direct injection of multiple mRNA species,provides for high protein expression and can accommodate the deleteriouseffects inherent in the overexpression of ENaC. The drawbacks arehowever that electrophysiological screening using amphibian oocytes isnot as amenable to high throughput screening of large numbers ofcompounds and is not a mammalian system. As noted, the present inventionembraces human delta beta gamma ENaC assays using mammalian cells,preferably high throughput assays.

ENaC proteins are known to form heteromeric channels comprised of threesubunits, an alpha, beta, and a gamma or delta subunit. The sequences ofthese respective ENaC subunits are disclosed in an earlier publishedpatent application by the present Assignee, U.S. Ser. No. 10/133,573which is incorporated by reference in its entirety herein. Additionally,the sequences for these respective subunits are contained in theSequence Listing that immediately precedes the claims of the subjectapplication. Upon co-expression in a suitable cell these subunits resultin a heterotrimeric channel having ion channel cation channel activity;in particular it responds to sodium and should similarly respond tolithium ions in cell-based assays such as those which are disclosedherein and in Senomyx's prior patent application referenced above.

Also different splice variants of these ENaC subunit sequences are knownwith some being the subject of recently filed provisional applicationsby the present Assignee. See also U.S. Pat. No. 5,693,756 incorporatedby reference in its entirety herein.

The ENaC channel has relatively high permeability to sodium and lithiumand is amiloride-sensitive. Channel activity can be effectivelymeasured, e.g., by recording ligand-induced changes in [Na+] andmeasuring sodium or lithium ion influx using fluorescent ion-indicatordyes and fluorimetric imaging. ENaC is expressed in a number ofepithelial tissues, including taste buds. Additionally, ENaC function isinvolved in kidney, lung function, blood pressure regulation et al. asdisclosed above. Consequently, compounds identified as ENaC modulatorshave significant potential human therapeutic applications.

The Senomyx application incorporated by reference provides highthroughput screening assays using mammalian cells transfected or seededinto wells or culture plates wherein functional expression in thepresence of test compounds is allowed to proceed and activity isdetected using membrane-potential fluorescent or ion (sodium)fluorescent dyes.

As discussed above, the invention specifically provides methods ofscreening for modulators, e.g., activators, inhibitors, stimulators,enhancers, etc., of human delta ENaC nucleic acids and proteins, usingthe human ENaC nucleic acid sequences provided herein. Such modulatorscan affect ENaC activity, e.g., by modulating ENaC transcription,translation, mRNA or protein stability; by altering the interaction ofENaC with the plasma membrane, or other molecules; or by affecting ENaCprotein activity. Compounds are screened, e.g., using high throughputscreening (HITS), to identify those compounds that can bind to and/ormodulate the activity of a ENaC polypeptide or fragment thereof. In thepresent invention, ENaC proteins are recombinantly expressed in cells,e.g., human cells, or frog oocytes and the modulation of ENaC is assayedby using any measure of ion channel function, such as measurement of themembrane potential, or measures of changes in intracellular sodium orlithium levels. Methods of assaying ion, e.g., cation, channel functioninclude, for example, patch clamp techniques, two electrode voltageclamping, measurement of whole cell currents, and fluorescent imagingtechniques that use ion-sensitive fluorescent dyes and ion flux assays,e.g., radiolabeled-ion flux assays or ion flux assays.

A human delta ENaC agonist identified as set forth in the currentapplication can be used for a number of different purposes. For example,a ENaC activator can be included as a flavoring agent to modulate thesalty taste of foods, beverages, soups, medicines, and other productsfor human consumption. Additionally, the invention provides kits forcarrying out the herein-disclosed assays.

Definitions

An ENaC associated biological function condition preferably refers tohuman salty taste perception.

“Cation channels” are a diverse group of proteins that regulate the flowof cations across cellular membranes. The ability of a specific cationchannel to transport particular cations typically varies with thevalency of the cations, as well as the specificity of the given channelfor a particular cation.

“Homomeric channel” refers to a cation channel composed of identicalalpha subunits, whereas “heteromeric channel” refers to a cation channelcomposed of two or more different types of alpha subunits. Bothhomomeric and heteromeric channels can include auxiliary beta subunits.

A “beta subunit” or “gamma subunit” is a polypeptide monomer that is anauxiliary subunit of a cation channel composed of alpha subunits;however, beta or gamma subunits alone cannot form a channel (see, e.g.,U.S. Pat. No. 5,776,734). Beta or gamma subunits are known, for example,to increase the number of channels by helping the alpha or deltasubunits reach the cell surface, change activation kinetics, and changethe sensitivity of natural ligands binding to the channels. Beta orgamma subunits can be outside of the pore region and associated withalpha or delta subunits comprising the pore region. They can alsocontribute to the external mouth of the pore region.

The term “authentic” or wild-type” or “native” human ENaC nucleic acidsequences refer to the wild-type and mutant alpha, beta, gamma and deltanucleic acid sequences contained in the Sequence Listing thatimmediately precedes the claims as well as splice variants and otherENaC nucleic acid sequences generally known in the art.

The term “authentic” or “wild-type” or “native” human ENaC polypeptidesrefers to the polypeptides encoded by the nucleic acid sequencecontained in SEQ ID NO: 1, 3 and 5 and contained in SEQ ID NO:2, 4 and6.

The term “modified hENaC nuclear acid sequence” or “optimized hENaCnucleic acid sequence” refers to a hENaC nucleic acid sequence whichcontains one or mutation that, particularly those that affect (inhibitor prevent) ENaC activity in recombinant host cells, and most especiallyoocytes or human cells such as HEK-293 cells. Particularly, thesemutations include those that affect gating by the resultant ENaC channelcontaining the mutated subunit sequence. The ENaC may comprise suchmutations in one or several of the three subunits that constitute theENaC. The modified ENaC nucleic acid sequence contains substitutionmutations in one subunit that affect (impair) gating function ordefective surface expression. The invention embraces the use of othermutated ENaC sequences, i.e., delta subunit mutants, e.g., splicevariants, those containing deletions or additions, chimeras of thesubject ENaC sequences and the like. Further, the invention may use ENaCsubunit sequences which may be modified to introduce host cell preferredcodons, particularly amphibian or human host cell preferred codons.

The term “ENaC” protein or fragment thereof, or a nucleic acid encoding“ENaC” or a fragment thereof refer to nucleic acids and polypeptidepolymorphic variants, alleles, mutants, and interspecies homologs that:(1) have an amino acid sequence that has greater than about 60% aminoacid sequence identity, 65%, 70%, 75%, 80%, 85%, 90%, preferably 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or greater amino acid sequenceidentity, preferably over a region of at least about 25, 50, 100, 200,500, 1000, or more amino acids, to an amino acid sequence encoded by aENaC nucleic acid or amino acid sequence of a ENaC protein, e.g., theENaC subunit proteins encoded by the ENaC nucleic acid sequencescontained in the Sequence Listing that precedes the claims of thisapplication as well as fragments thereof, and conservatively modifiedvariants thereof; (3) polypeptides encoded by nucleic acid sequenceswhich specifically hybridize under stringent hybridization conditions toan anti-sense strand corresponding to a nucleic acid sequence encoding aENaC protein subunit, and conservatively modified variants thereof; (4)have a nucleic acid sequence that has greater than about 60% sequenceidentity, 65%, 70%, 75%, 80%, 85%, 90%, preferably 91%, 92%, 93%, 94%,95%, 96%, 97%, 98% or 99%, or higher nucleotide sequence identity,preferably over a region of at least about 25, 50, 100, 200, 500, 1000,or more nucleotides, to a ENaC nucleic acid, e.g., those disclosedherein.

An ENaC polynucleotide or polypeptide sequence is typically from amammal including, but not limited to, primate, e.g., human; rodent,e.g., rat, mouse, hamster; cow, pig, horse, sheep, or any mammal. Thenucleic acids and proteins of the invention include both naturallyoccurring or recombinant molecules. ENaC proteins typically have ionchannel activity, i.e., they are permeable to sodium or lithium.

By “determining the functional effect” or “determining the effect on thecell” is meant assaying the effect of a compound that increases ordecreases a parameter that is indirectly or directly under the influenceof a ENaC polypeptide e.g., functional, physical, phenotypic, andchemical effects. Such functional effects include, but are not limitedto, changes in ion flux, membrane potential, current amplitude, andvoltage gating, as well as other biological effects such as changes ingene expression of ENaC or of any marker genes, and the like. The ionflux can include any ion that passes through the channel, e.g., sodiumor lithium, and analogs thereof such as radioisotopes. Such functionaleffects can be measured by any means known to those skilled in the art,e.g., patch clamping, using voltage-sensitive dyes, or by measuringchanges in parameters such as spectroscopic characteristics (e.g.,fluorescence, absorbance, refractive index), hydrodynamic (e.g., shape),chromatographic, or solubility properties.

“Inhibitors,” “activators,” and “modulators” of ENaC polynucleotide andpolypeptide sequences are used to refer to activating, inhibitory, ormodulating molecules identified using in vitro and in vivo assays ofENaC polynucleotide and polypeptide sequences. Inhibitors are compoundsthat, e.g., bind to, partially or totally block activity, decrease,prevent, delay activation, inactivate, desensitize, or down regulate theactivity or expression of ENaC proteins, e.g., antagonists.

“Activators” are compounds that increase, open, activate, facilitate,enhance activation, sensitize, agonize, or up regulate ENaC proteinactivity. Inhibitors, activators, or modulators also include geneticallymodified versions of ENaC proteins, e.g., versions with alteredactivity, as well as naturally occurring and synthetic ligands,antagonists, agonists, peptides, cyclic peptides, nucleic acids,antibodies, antisense molecules, siRNA, ribozymes, small organicmolecules and the like. Such assays for inhibitors and activatorsinclude, e.g., expressing ENaC protein in vitro, in cells, cellextracts, or cell membranes, applying putative modulator compounds, andthen determining the functional effects on activity, as described above.

1 Samples or assays comprising ENaC proteins that are treated with apotential activator, inhibitor, or modulator are compared to controlsamples without the inhibitor, activator, or modulator to examine theextent of activation or migration modulation. Control samples (untreatedwith inhibitors) are assigned a relative protein activity value of 100%.Inhibition of ENaC is achieved when the activity value relative to thecontrol is about 80%, preferably 50%, more preferably 25-0%. Activationof ENaC is achieved when the activity value relative to the control(untreated with activators) is 110%, more preferably 150%, morepreferably 200-500% (i.e., two to five fold higher relative to thecontrol), more preferably 1000-3000% higher.

The term “test compound” or “drug candidate” or “modulator” orgrammatical equivalents as used herein describes any molecule, eithernaturally occurring or synthetic compound, preferably a small molecule,or a protein, oligopeptide (e.g., from about 5 to about 25 amino acidsin length, preferably from about 10 to 20 or 12 to 18 amino acids inlength, preferably 12, 15, or 18 amino acids in length), small organicmolecule, polysaccharide, lipid, fatty acid, polynucleotide, siRNA,oligonucleotide, ribozyme, etc., to be tested for the capacity tomodulate ENaC function. The test compound can be in the form of alibrary of test compounds, such as a combinatorial or randomized librarythat provides a sufficient range of diversity. Test compounds areoptionally linked to a fusion partner, e.g., targeting compounds, rescuecompounds, dimerization compounds, stabilizing compounds, addressablecompounds, and other functional moieties. Conventionally, new chemicalentities with useful properties are generated by identifying a testcompound (called a “lead compound”) with some desirable property oractivity, e.g., inhibiting activity, creating variants of the leadcompound, and evaluating the property and activity of those variantcompounds. Often, high throughput screening (HTS) methods are employedfor such an analysis.

A “small organic molecule” refers to an organic molecule, eithernaturally occurring or synthetic, that has a molecular weight of morethan about 50 daltons and less than about 2500 daltons, preferably lessthan about 2000 daltons, preferably between about 100 to about 1000daltons, more preferably between about 200 to about 500 daltons.

“Biological sample” include sections of tissues such as biopsy andautopsy samples, and frozen sections taken for histologic purposes. Suchsamples include blood, sputum, tissue, cultured cells, e.g., primarycultures, explants, and transformed cells, stool, urine, etc. Abiological sample is typically obtained from a eukaryotic organism, mostpreferably a mammal such as a primate e.g., chimpanzee or human; cow;dog; cat; a rodent, e.g., guinea pig, rat, mouse; rabbit; or a bird;reptile; or fish.

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same(i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over aspecified region (e.g., ENaC nucleotide sequences contained in theSequence Listing), when compared and aligned for maximum correspondenceover a comparison window or designated region) as measured using a BLASTor BLAST 2.0 sequence comparison algorithms with default parametersdescribed below, or by manual alignment and visual inspection (see,e.g., NCBI web site or the like). Such sequences are then said to be“substantially identical.” This definition also refers to, or may beapplied to, the compliment of a test sequence. The definition alsoincludes sequences that have deletions and/or additions, as well asthose that have substitutions. As described below, the preferredalgorithms can account for gaps and the like. Preferably, identityexists over a region that is at least about 25 amino acids ornucleotides in length, or more preferably over a region that is 50-100amino acids or nucleotides in length.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Preferably,default program parameters can be used, or alternative parameters can bedesignated. The sequence comparison algorithm then calculates thepercent sequence identities for the test sequences relative to thereference sequence, based on the program parameters.

A “comparison window”, as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of from 20 to 600, usually about 50 to about 200, moreusually about 100 to about 150 in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Methods of alignment of sequencesfor comparison are well-known in the art. Optimal alignment of sequencesfor comparison can be conducted, e.g., by the local homology algorithmof Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homologyalignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970),by the search for similarity method of Pearson & Lipman, Proc. Nat'l.Acad. Sci. USA 85:2444 (1988), by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Dr., Madison,Wis.), or by manual alignment and visual inspection (see, e.g., CurrentProtocols in Molecular Biology (Ausubel et al., eds. 1995 supplement)).

A preferred example of algorithm that is suitable for determiningpercent sequence identity and sequence similarity are the BLAST andBLAST 2.0 algorithms, which are described in Altschul et al., Nucl.Acids Res. 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol.215:403-410 (1990), respectively. BLAST and BLAST 2.0 are used, with theparameters described herein, to determine percent sequence identity forthe nucleic acids and proteins of the invention. Software for performingBLAST analyses is publicly available through the National Center forBiotechnology Information. This algorithm involves first identifyinghigh scoring sequence pairs (HSPs) by identifying short words of lengthW in the query sequence, which either match or satisfy somepositive-valued threshold score T when aligned with a word of the samelength in a database sequence. T is referred to as the neighborhood wordscore threshold (Altschul et al., supra). These initial neighborhoodword hits act as seeds for initiating searches to find longer HSPscontaining them. The word hits are extended in both directions alongeach sequence for as far as the cumulative alignment score can beincreased. Cumulative scores are calculated using, for nucleotidesequences, the parameters M (reward score for a pair of matchingresidues; always>0) and N (penalty score for mismatching residues;always<0). For amino acid sequences, a scoring matrix is used tccalculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a word length (W) of 11, anexpectation (E) of 10, M=5, N=−4 and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a word lengthof 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff & Henikoff, Proc. Natl. Acad. Sci., USA 89:10915 (1989))alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparisonof both strands.

“Nucleic acid” refers to deoxyribonucleotides or ribonucleotides andpolymers thereof in either single- or double-stranded form, andcomplements thereof. The term encompasses nucleic acids containing knownnucleotide analogs or modified backbone residues or linkages, which aresynthetic, naturally occurring, and non-naturally occurring, which havesimilar binding properties as the reference nucleic acid and which aremetabolized in a manner similar to the reference nucleotides. Examplesof such analogs include, without limitation, phosphorothioates,phosphoramidates, methyl phosphonates, chiral-methyl phosphonates,2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs).

Unless otherwise indicated, a particular nucleic acid sequence alsoimplicitly encompasses conservatively modified variants thereof (e.g.,degenerate codor substitutions) and complementary sequences, as well asthe sequence explicitly indicated. Specifically, degenerate codonsubstitutions may be achieved by generating sequences in which the thirdposition of one or more selected (or all) codons is substituted withmixed-base and/or deoxyinosine residues (Batzer et al., Nucleic AcidRes. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608(1985); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). The termnucleic acid is used interchangeably with gene, cDNA, mRNA,oligonucleotide, and polynucleotide.

A particular nucleic acid sequence also implicitly encompasses “splicevariants.” Similarly, a particular protein encoded by a nucleic acidimplicitly encompasses any protein encoded by a splice variant of thatnucleic acid. “Splice variants,” as the name suggests, are products ofalternative splicing of a gene. After transcription, an initial nucleicacid transcript may be spliced such that different (alternate) nucleicacid splice products encode different polypeptides. Mechanisms for theproduction of splice variants vary, but include alternate splicing ofexons. Alternate polypeptides derived from the same nucleic acid byread-through transcription are also encompassed by this definition. Anyproducts of a splicing reaction, including recombinant forms of thesplice products, are included in this definition. An example ofpotassium channel splice variants is discussed in Leicher, et al., J.Biol. Chem. 273(52):35095-35101 (1998).

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers and non-naturally occurring amino acid polymer.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an a carbon that is bound toa hydrogen, a carboxyl group, an amino group, and an R group, e.g.,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Amino acid mimetics refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that functions in amanner similar to a naturally occurring amino acid.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes.

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, conservatively modified variants refers to those nucleicacids which encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences. Because of the degeneracyof the genetic code, a large number of functionally identical nucleicacids encode any given protein. For instance, the codons GCA, GCC, GCGand GCU all encode the amino acid alanine. Thus, at every position wherean alanine is specified by a codon, the codon can be altered to any ofthe corresponding codons described without altering the encodedpolypeptide. Such nucleic acid variations are “silent variations,” whichare one species of conservatively modified variations. Every nucleicacid sequence herein which encodes a polypeptide also describes everypossible silent variation of the nucleic acid. One of skill willrecognize that each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine, and TGG, which is ordinarilythe only codon for tryptophan) can be modified to yield a functionallyidentical molecule. Accordingly, each silent variation of a nucleic acidwhich encodes a polypeptide is implicit in each described sequence withrespect to the expression product, but not with respect to actual probesequences.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant” where the alteration results inthe substitution of an amino acid with a chemically similar amino acid.Conservative substitution tables providing functionally similar aminoacids are well known in the art. Such conservatively modified variantsare in addition to and do not exclude polymorphic variants, interspecieshomologs, and alleles of the invention.

The following eight groups each contain amino acids that areconservative substitutions for one another: 1) Alanine (A), Glycine (G);2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine(Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L),Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y),Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C),Methionine (M) (see, e.g., Creighton, Proteins (1984)).

Macromolecular structures such as polypeptide structures can bedescribed in terms of various levels of organization. For a generaldiscussion of this organization, see, e.g., Alberts et al., MolecularBiology of the Cell (3^(rd) ed., 1994) and Cantor and Schimmel,Biophysical Chemistry Part I: The Conformation of BiologicalMacromolecules (1980). “Primary structure” refers to the amino acidsequence of a particular peptide. “Secondary structure” refers tolocally ordered, three dimensional structures within a polypeptide.These structures are commonly known as domains, e.g., transmembranedomains, pore domains, and cytoplasmic tail domains. Domains areportions of a polypeptide that form a compact unit of the polypeptideand are typically 15 to 350 amino acids long. Exemplary domains includeextracellular domains, transmembrane domains, and cytoplasmic domains.Typical domains are made up of sections of lesser organization such asstretches of .beta.-sheet and .alpha.-helices. “Tertiary structure”refers to the complete three dimensional structure of a polypeptidemonomer. “Quaternary structure” refers to the three dimensionalstructure formed by the noncovalent association of independent tertiaryunits. Anisotropic terms are also known as energy terms.

A “label” or a “detectable moiety” is a composition detectable byspectroscopic, photochemical, biochemical, immunochemical, chemical, orother physical means. For example, useful labels include ³²P,fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonlyused in an ELISA), biotin, digoxigenin, or haptens and proteins whichcan be made detectable, e.g., by incorporating a radiolabel into thepeptide or used to detect antibodies specifically reactive with thepeptide.

The term “recombinant” when used with reference, e.g., to a cell, ornucleic acid, protein, or vector, indicates that the cell, nucleic acid,protein or vector, has been modified by the introduction of aheterologous nucleic acid or protein or the alteration of a nativenucleic acid or protein, or that the cell is derived from a cell somodified. Thus, for example, recombinant cells express genes that arenot found within the native (non-recombinant) form of the cell orexpress native genes that are otherwise abnormally expressed, underexpressed or not expressed at all.

The term “heterologous” when used with reference to portions of anucleic acid indicates that the nucleic acid comprises two or moresubsequences that are not found in the same relationship to each otherin nature. For instance, the nucleic acid is typically recombinantlyproduced, having two or more sequences from unrelated genes arranged tomake a new functional nucleic acid, e.g., a promoter from one source anda coding region from another source. Similarly, a heterologous proteinindicates that the protein comprises two or more subsequences that arenot found in the same relationship to each other in nature (e.g., afusion protein).

The phrase “stringent hybridization conditions” refers to conditionsunder which a probe will hybridize to its target subsequence, typicallyin a complex mixture of nucleic acids, but to no other sequences.Stringent conditions are sequence-dependent and will be different indifferent circumstances. Longer sequences hybridize specifically athigher temperatures. An extensive guide to the hybridization of nucleicacids is found in Tijssen, Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Probes, “Overview of principles ofhybridization and the strategy of nucleic acid assays” (1993).Generally, stringent conditions are selected to be about 5-10° C. lowerthan the thermal melting point (T_(m)) for the specific sequence at adefined ionic strength pH. The T_(m) is the temperature (under definedionic strength, pH, and nucleic concentration) at which 50% of theprobes complementary to the target hybridize to the target sequence atequilibrium (as the target sequences are present in excess, at T_(m),50% of the probes are occupied at equilibrium). Stringent conditions mayalso be achieved with the addition of destabilizing agents such asformamide. For selective or specific hybridization, a positive signal isat least two times background, preferably 10 times backgroundhybridization. Exemplary stringent hybridization conditions can be asfollowing: 50% formamide, 5×SSC, and 1% SDS, incubating at 42° C., or,5×SSC, 1% SDS, incubating at 65° C., with wash in 0.2.times. SSC, and0.1% SDS at 65° C.

Nucleic acids that do not hybridize to each other under stringentconditions are still substantially identical if the polypeptides whichthey encode are substantially identical. This occurs, for example, whena copy of a nucleic acid is created using the maximum codon degeneracypermitted by the genetic code. In such cases, the nucleic acidstypically hybridize under moderately stringent hybridization conditions.Exemplary “moderately stringent hybridization conditions” include ahybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37° C.,and a wash in 1.times. SSC at 45° C. A positive hybridization is atleast twice background. Those of ordinary skill will readily recognizethat alternative hybridization and wash conditions can be utilized toprovide conditions of similar stringency. Additional guidelines fordetermining hybridization parameters are provided in numerous reference,e.g., and Current Protocols in Molecular Biology, ed. Ausubel, et al.

For PCR, a temperature of about 36° C. is typical for low stringencyamplification, although annealing temperatures may vary between about32° C. and 48° C. depending on primer length. For high stringency PCRamplification, a temperature of about 62° C. is typical, although highstringency annealing temperatures can range from about 50° C. to about65° C., depending on the primer length and specificity. Typical cycleconditions for both high and low stringency amplifications include adenaturation phase of 90° C.−95° C. for 30 sec-2 min., an annealingphase lasting 30 sec.-2 min., and an extension phase of about 72° C. for1-2 min. Protocols and guidelines for low and high stringencyamplification reactions are provided, e.g., in Innis et al. (1990) PCRProtocols, A Guide to Methods and Applications, Academic Press, Inc.N.Y.).

1 “Antibody” refers to a polypeptide comprising a framework region froman immunoglobulin gene or fragments thereof that specifically binds andrecognizes an antigen. The recognized immunoglobulin genes include thekappa, lambda, alpha, gamma, delta, epsilon, and mu constant regiongenes, as well as the myriad immunoglobulin variable region genes. Lightchains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, which in turn definethe immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.Typically, the antigen-binding region of an antibody will be mostcritical in specificity and affinity of binding.

The term antibody, as used herein, also includes antibody fragmentseither produced by the modification of whole antibodies, or thosesynthesized de novo using recombinant DNA methodologies (e.g., singlechain Fv), chimeric, humanized or those identified using phage displaylibraries (see, e.g., McCafferty et al., Nature 348:552-554 (1990)) Forpreparation of antibodies, e.g., recombinant, monoclonal, or polyclonalantibodies, many technique known in the art can be used (see, e.g.,Kohler & Milstein, Nature 256:495-497 (1975); Kozbor et al., ImmunologyToday 4: 72 (1983); Cole et al., pp. 77-96 in Monoclonal Antibodies andCancer Therapy, Alan R. Liss, Inc. (1985); Coligan, Current Protocols inImmunology (1991); Harlow & Lane, Antibodies, A Laboratory Manual (1988)and Harlow & Lane, Using Antibodies, A Laboratory Manual (1999); andGoding, Monoclonal Antibodies: Principles and Practice (2d ed. 1986)).

The phrase “specifically (or selectively) binds” to an antibody or“specifically (or selectively) immunoreactive with,” when referring to aprotein or peptide, refers to a binding reaction that is determinativeof the presence of the protein, often in a heterogeneous population ofproteins and other biologics. Thus, under designated immunoassayconditions, the specified antibodies bind to a particular protein atleast two times the background and more typically more than 10 to 100times background. Specific binding to an antibody under such conditionsrequires an antibody that is selected for its specificity for aparticular protein. For example, polyclonal antibodies raised to ENaCprotein, polymorphic variants, alleles, orthologs, and conservativelymodified variants, or splice variants, or portions thereof, can beselected to obtain only those polyclonal antibodies that arespecifically immunoreactive with ENaC proteins and not with otherproteins. This selection may be achieved by subtracting out antibodiesthat cross-react with other molecules. A variety of immunoassay formatsmay be used to select antibodies specifically immunoreactive with aparticular protein. For example, solid-phase ELISA immunoassays areroutinely used to select antibodies specifically immunoreactive with aprotein (see, e.g., Harlow & Lane, Antibodies, A Laboratory Manual(1988) for a description of immunoassay formats and conditions that canbe used to determine specific immunoreactivity).

By “therapeutically effective dose” herein is meant a dose that produceseffects for which it is administered. The exact dose will depend on thepurpose of the treatment, and will be ascertainable by one skilled inthe art using known techniques (see, e.g., Lieberman, PharmaceuticalDosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technologyof Pharmaceutical Compounding (1999); and Pickar, Dosage Calculations(1999)).

Recombinant Expression of ENaC

To obtain high level expression of a cloned gene, such as those cDNAsencoding ENaC, one typically subclones ENaC into an expression vectorthat contains a strong promoter to direct transcription, atranscription/translation terminator, and if for a nucleic acid encodinga protein, a ribosome binding site for translational initiation.Suitable eukaryotic and prokaryotic promoters are well known in the artand described, e.g., in Sambrook et al., and Ausubel et al., supra. Forexample, bacterial expression systems for expressing the ENaC proteinare available in, e.g., E. coli, Bacillus sp., and Salmonella (Palva etal., Gene 22:229-235 (1983); Mosbach et al., Nature 302:543-545 (1983).Kits for such expression systems are commercially available. Eukaryoticexpression systems for mammalian cells, yeast, and insect cells are wellknown in the art and are also commercially available. For example,retroviral expression systems may be used in the present invention. Asdescribed infra, the subject modified hENaC is preferably expressed inhuman cells such as HEK-293 cells which are widely used for highthroughput screening.

Selection of the promoter used to direct expression of a heterologousnucleic acid depends on the particular application. The promoter ispreferably positioned about the same distance from the heterologoustranscription start site as it is from the transcription start site inits natural setting. As is known in the art, however, some variation inthis distance can be accommodated without loss of promoter function.

In addition to the promoter, the expression vector typically contains atranscription unit or expression cassette that contains all theadditional elements required for the expression of the ENaC-encodingnucleic acid in host cells. A typical expression cassette thus containsa promoter operably linked to the nucleic acid sequence encoding ENaCand signals required for efficient polyadenylation of the transcript,ribosome binding sites, and translation termination. Additional elementsof the cassette may include enhancers and, if genomic DNA is used as thestructural gene, introns with functional splice donor and acceptorsites. As noted previously, the exemplified modified hENaC is modifiedto remove putative cryptic splice donor and acceptor sites.

In addition to a promoter sequence, the expression cassette should alsocontain a transcription termination region downstream of the structuralgene to provide for efficient termination. The termination region may beobtained from the same gene as the promoter sequence or may be obtainedfrom different genes.

The particular expression vector used to transport the geneticinformation into the cell is not particularly critical. Any of theconventional vectors used for expression in eukaryotic or prokaryoticcells may be used. Standard bacterial expression vectors includeplasmids such as pBR322 based plasmids, pSKF, pET23D, and fusionexpression systems such as MBP, GST, and LacZ. Epitope tags can also beadded to recombinant proteins to provide convenient methods ofisolation, e.g., c-myc. Sequence tags may be included in an expressioncassette for nucleic acid rescue. Markers such as fluorescent proteins,green or red fluorescent protein, Bf-gal, CAT, and the like can beincluded in the vectors as markers for vector transduction.

Expression vectors containing regulatory elements from eukaryoticviruses are typically used in eukaryotic expression vectors, e.g., SV40vectors, papilloma virus vectors, retroviral vectors, and vectorsderived from Epstein-Barr virus. Other exemplary eukaryotic vectorsinclude pMSG, pAV009/A⁺, pMTO10/A⁺, pMAMneo-5, baculovirus pDSVE, andany other vector allowing expression of proteins under the direction ofthe CMV promoter, SV40 early promoter, SV40 later promoter,metallothionein promoter, murine mammary tumor virus promoter, Roussarcoma virus promoter, polyhedrin promoter, or other promoters showneffective for expression in eukaryotic cells.

Expression of proteins from eukaryotic vectors can be also be regulatedusing inducible promoters. With inducible promoters, expression levelsare tied to the concentration of inducing agents, such as tetracyclineor ecdysone, by the incorporation of response elements for these agentsinto the promoter. Generally, high level expression is obtained frominducible promoters only in the presence of the inducing agent; basalexpression levels are minimal.

The vectors used in the invention may include a regulatable promoter,e.g., tet-regulated systems and the RU-486 system (see, e.g., Gossen &Bujard, Proc. Nat'l Acad. Sci USA 89:5547 (1992); Oligino et al., GeneTher. 5:491-496 (1998); Wang et al., Gene Ther. 4:432-441 (1997);Neering et al., Blood 88:1147-1155 (1996); and Rendahl et al., Nat.Biotechnol. 16:757-761 (1998)). These impart small molecule control onthe expression of the candidate target nucleic acids. This beneficialfeature can be used to determine that a desired phenotype is caused by atransfected cDNA rather than a somatic mutation.

Some expression systems have markers that provide gene amplificationsuch as thymidine kinase and dihydrofolate reductase. Alternatively,high yield expression systems not involving gene amplification are alsosuitable, such as using a baculovirus vector in insect cells, with aENaC encoding sequence under the direction of the polyhedrin promoter orother strong baculovirus promoters.

The elements that are typically included in expression vectors alsoinclude a replicon that functions in the particular host cell. In thecase of E. coli, the vector may contain a gene encoding antibioticresistance to permit selection of bacteria that harbor recombinantplasmids, and unique restriction sites in nonessential regions of theplasmid to allow insertion of eukaryotic sequences. The particularantibiotic resistance gene chosen is not critical, any of the manyresistance genes known in the art are suitable. The prokaryoticsequences are preferably chosen such that they do not interfere with thereplication of the DNA in eukaryotic cells, if necessary.

Standard transfection methods may be used to produce bacterial,mammalian, yeast or insect cell lines that express large quantities ofENaC protein, which are then purified using standard techniques (see,e.g., Colley et al., J. Biol. Chem. 264:17619-17622 (1989); Guide toProtein Purification, in Methods in Enzymology, Vol. 182 (Deutscher,ed., 1990)). Transformation of eukaryotic and prokaryotic cells areperformed according to standard techniques (see, e.g., Morrison, J.Bact. 132:349-351 (1977); Clark-Curtiss & Curtiss, Methods in Enzymology101:347-362 (Wu et al., eds, 1983). Any of the well-known procedures forintroducing foreign nucleotide sequences into host cells may be used.These include the use of calcium phosphate transfection, polybrene,protoplast fusion, electroporation, biolistics, liposomes,microinjection, plasma vectors, viral vectors and any of the other wellknown methods for introducing cloned genomic DNA, cDNA, synthetic DNA orother foreign genetic material into a host cell (see, e.g., Sambrook etal., supra). It is only necessary that the particular geneticengineering procedure used be capable of successfully introducing atleast one gene into the host cell capable of expressing ENaC.

After the expression vector is introduced into the cells, thetransfected cells are cultured under conditions favoring expression ofENaC. In some instances, such ENaC polypeptides may be recovered fromthe culture using standard techniques identified below.

Assays for Modulators of Delta ENaC Protein

Modulation of an ENaC protein, can be assessed using a variety of invitro and in vivo assays, including cell-based models as describedabove. Such assays can be used to test for inhibitors and activators ofENaC protein or fragments thereof, and, consequently, inhibitors andactivators of ENaC. Such modulators of ENaC protein are useful asflavorings to modulate ENaC associated salty taste.

As noted above, the ENaC protein used in the subject cell based assayswill preferably be encoded by hENaC nucleic acid sequences encodingsubunits that comprise at least one mutation which affects (reduces)ENaC function relative to the corresponding wild-type ENaC as the assayspreferably screen for compounds (enhancers) capable of “restoring” thefunction thereof in specific cells, preferably frog oocytes or mammaliancells, preferably human cells. These sequences include those exemplifiedin the examples infra.

Compounds identified in such assays will then be evaluated in vivo toconfirm that this affect on ENaC is obtained in vivo and consequentlythat the identified compound is suitable for correcting or modulating afunction involving ENaC such as those afore-identified. Assays usingcells expressing ENaC proteins, either recombinant or naturallyoccurring, can be performed using a variety of assays, in vitro, invivo, and ex vivo, as described herein. To identify molecules capable ofmodulating ENaC, assays are performed to detect the effect of variouscandidate modulators on ENaC activity preferably a mutant ENaC in acell.

The channel activity of ENaC proteins can be assayed using a variety ofassays to measure changes in ion fluxes including patch clamptechniques, measurement of whole cell currents, radiolabeled ion fluxassays or a flux assay coupled to atomic absorption spectroscopy, andfluorescence assays using voltage-sensitive dyes or lithium or sodiumsensitive dyes (see, e.g., Vestergarrd-Bogind et al., J. Membrane Biol.88:67-75 (1988); Daniel et al., J. Pharmacol. Meth. 25:185-193 (1991);Hoevinsky et al., J. Membrane Biol. 137:59-70 (1994)). For example, anucleic acid encoding an ENaC protein or homolog thereof can be injectedinto Xenopus oocytes or transfected into mammalian cells, preferablyhuman cells such as HEK-293 cells. Channel activity can then be assessedby measuring changes in membrane polarization, i.e., changes in membranepotential.

A preferred means to obtain electrophysiological measurements is bymeasuring currents using patch clamp techniques, e.g., the“cell-attached” mode, the “inside-out” mode, and the “whole cell” mode(see, e.g., Ackerman et al., New Engl. J. Med. 336:1575-1595, 1997).Whole cell currents can be determined using standard methodology such asthat described by Hamil et al., Pflugers. Archiv. 391:185 (1981).

Channel activity is also conveniently assessed by measuring changes inintracellular ion levels, i.e., sodium or lithium. Such methods areexemplified herein. For example, sodium flux can be measured byassessment of the uptake of radiolabeled sodium or by using suitablefluorescent dyes. In a typical microfluorimetry assay, a dye whichundergoes a change in fluorescence upon binding a single sodium ion, isloaded into the cytosol of ENaC-expressing cells. Upon exposure to ENaCagonist, an increase in cytosolic sodium is reflected by a change influorescence that occurs when sodium is bound.

The activity of ENaC polypeptides can in addition to these preferredmethods also be assessed using a variety of other in vitro and in vivoassays to determine functional, chemical, and physical effects, e.g.,measuring the binding of ENaC to other molecules, including peptides,small organic molecules, and lipids; measuring ENaC protein and/or RNAlevels, or measuring other aspects of ENaC polypeptides, e.g.,transcription levels, or physiological changes that affects ENaCactivity. When the functional consequences are determined using intactcells or animals, one can also measure a variety of effects such aschanges in cell growth or pH changes or changes in intracellular secondmessengers such as IP3, cGMP, or cAMP, or components or regulators ofthe phospholipase C signaling pathway. Such assays can be used to testfor both activators and inhibitors of ENaC proteins. Modulators thusidentified are useful for, e.g., many diagnostic and therapeuticapplications.

In Vitro Assays

Assays to identify compounds with ENaC modulating activity arepreferably performed in vitro. The assays herein preferably use fulllength ENaC protein or a variant thereof. This protein can optionally befused to a heterologous protein to form a chimera. In the assaysexemplified herein, cells which express the full-length ENaC polypeptideare used in high throughput assays are used to identify compounds thatmodulate wild-type and mutant ENaCs. Alternatively, purified recombinantor naturally occurring ENaC protein can be used in the in vitro methodsof the invention. In addition to purified ENaC protein or fragmentthereof, the recombinant or naturally occurring ENaC protein can be partof a cellular lysate or a cell membrane. As described below, the bindingassay can be either solid state or soluble. Preferably, the protein,fragment thereof or membrane is bound to a solid support, eithercovalently or non-covalently. Often, the in vitro assays of theinvention are ligand binding or ligand affinity assays, eithernon-competitive or competitive (with known extracellular ligands such asmenthol). Other in vitro assays include measuring changes inspectroscopic (e.g., fluorescence, absorbance, refractive index),hydrodynamic (e.g., shape), chromatographic, or solubility propertiesfor the protein.

Preferably, a high throughput binding assay is performed in which theENaC protein is contacted with a potential modulator and incubated for asuitable amount of time. A wide variety of modulators can be used, asdescribed below, including small organic molecules, peptides,antibodies, and ENaC ligand analogs. A wide variety of assays can beused to identify ENaC-modulator binding, including labeledprotein-protein binding assays, electrophoretic mobility shifts,immunoassays, enzymatic assays such as phosphorylation assays, and thelike. In some cases, the binding of the candidate modulator isdetermined through the use of competitive binding assays, whereinterference with binding of a known ligand is measured in the presenceof a potential modulator. Ligands for the ENaC family are known. Alsoamiloride and phenamil are known to inhibit ENaC function. In suchassays the known ligand is bound first, and then the desired compoundi.e., putative enhancer is added. After the ENaC protein is washed,interference with binding, either of the potential modulator or of theknown ligand, is determined. Often, either the potential modulator orthe known ligand is labeled.

In addition, high throughput functional genomics assays can also be usedto identify modulators of cold sensation by identifying compounds thatdisrupt protein interactions between ENaC and other proteins to which itbinds. Such assays can, e.g., monitor changes in cell surface markerexpression, changes in intracellular calcium, or changes in membranecurrents using either cell lines or primary cells. Typically, the cellsare contacted with a cDNA or a random peptide library (encoded bynucleic acids). The cDNA library can comprise sense, antisense, fulllength, and truncated cDNAs. The peptide library is encoded by nucleicacids. The effect of the cDNA or peptide library on the phenotype of thecells is then monitored, using an assay as described above. The effectof the cDNA or peptide can be validated and distinguished from somaticmutations, using, e.g., regulatable expression of the nucleic acid suchas expression from a tetracycline promoter. cDNAs and nucleic acidsencoding peptides can be rescued using techniques known to those ofskill in the art, e.g., using a sequence tag.

Proteins interacting with the ENaC protein encoded by the cDNA can beisolated using a yeast two-hybrid system, mammalian two hybrid system,or phage display screen, etc. Targets so identified can be further usedas bait in these assays to identify additional components that mayinteract with the ENaC channel which members are also targets for drugdevelopment (see, e.g., Fields et al., Nature 340:245 (1989); Vasavadaet al., Proc. Nat'l Acad. Sci. USA 88:10686 (1991); Fearon et al., Proc.Nat'l Acad. Sci. USA 89:7958 (1992); Dang et al., Mol. Cell. Biol.11:954 (1991); Chien et al., Proc. Nat'l Acad. Sci. USA 9578 (1991); andU.S. Pat. Nos. 5,283,173, 5,667,973, 5,468,614, 5,525,490, and5,637,463).

Cell-Based In Vivo Assays

In preferred embodiments, wild-type and mutant ENaC subunit proteins areexpressed in a cell, and functional, e.g., physical and chemical orphenotypic, changes are assayed to identify ENaC modulators thatmodulate ENaC function or which restore the function of mutant ENaCs,e.g., those having impaired gating function. Cells expressing ENaCproteins can also be used in binding assays. Any suitable functionaleffect can be measured, as described herein. For example, changes inmembrane potential, changes in intracellular lithium or sodium levels,and ligand binding are all suitable assays to identify potentialmodulators using a cell based system. Suitable cells for such cell basedassays include both primary cells and recombinant cell lines engineeredto express a ENaC protein. The ENaC proteins therefore can be naturallyoccurring or recombinant. Also, as described above, fragments of ENaCproteins or chimeras with ion channel activity can be used in cell basedassays. For example, a transmembrane domain of a ENaC protein can befused to a cytoplasmic domain of a heterologous protein, preferably aheterologous ion channel protein. Such a chimeric protein would have ionchannel activity and could be used in cell based assays of theinvention. In another embodiment, a domain of the ENaC protein, such asthe extracellular or cytoplasmic domain, is used in the cell-basedassays of the invention.

In another embodiment, cellular ENaC polypeptide levels can bedetermined by measuring the level of protein or mRNA. The level of ENaCprotein or proteins related to ENaC ion channel activation are measuredusing immunoassays such as western blotting, ELISA and the like with anantibody that selectively binds to the ENaC polypeptide or a fragmentthereof. For measurement of mRNA, amplification, e.g., using PCR, LCR,or hybridization assays, e.g., northern hybridization, RNAse protection,dot blotting, are preferred. The level of protein or mRNA is detectedusing directly or indirectly labeled detection agents, e.g.,fluorescently or radioactively labeled nucleic acids, radioactively orenzymatically labeled antibodies, and the like, as described herein.

Alternatively, ENaC expression can be measured using a reporter genesystem. Such a system can be devised using a ENaC protein promoteroperably linked to a reporter gene such as chloramphenicolacetyltransferase, firefly luciferase, bacterial luciferase,β-galactosidase and alkaline phosphatase. Furthermore, the protein ofinterest can be used as an indirect reporter via attachment to a secondreporter such as red or green fluorescent protein (see, e.g., Mistili &Spector, Nature Biotechnology 15:961-964 (1997)). The reporter constructis typically transfected into a cell. After treatment with a potentialmodulator, the amount of reporter gene transcription, translation, oractivity is measured according to standard techniques known to those ofskill in the art.

In another embodiment, a functional effect related to signaltransduction can be measured. An activated or inhibited ENaC will alterthe properties of target enzymes, second messengers, channels, and othereffector proteins. The examples include the activation of phospholipaseC and other signaling systems. Downstream consequences can also beexamined such as generation of diacyl glycerol and IP3 by phospholipaseC.

Assays for ENaC activity include cells that are loaded with ion orvoltage sensitive dyes to report activity, e.g., by observing sodiuminflux or intracellular sodium release. Assays for determining activityof such receptors can also use known agonists and antagonists for ENaCreceptors as negative or positive controls to assess activity of testedcompounds. In assays for identifying modulatory compounds (e.g.,agonists, antagonists), changes in the level of ions in the cytoplasm ormembrane voltage will be monitored using an ion sensitive or membranevoltage fluorescent indicator, respectively. Among the ion-sensitiveindicators and voltage probes that may be employed are those disclosedin the Molecular Probes 1997 Catalog. Radiolabeled ion flux assays or aflux assay coupled to atomic absorption spectroscopy can also be used.

Animal Models

Animal models also find potential use in screening for modulators ofENaC activity. Similarly, transgenic animal technology including geneknockout technology, for example as a result of homologous recombinationwith an appropriate gene targeting vector, or gene overexpression, willresult in the absence or increased expression of the ENaC protein. Thesame technology can also be applied to make knock-out cells. Whendesired, tissue-specific expression or knockout of the ENaC protein maybe necessary. Transgenic animals generated by such methods find use asanimal models of ENaC related responses.

Knock-out cells and transgenic mice can be made by insertion of a markergene or other heterologous gene into an endogenous ENaC gene site in themouse genome via homologous recombination. Such mice can also be made bysubstituting an endogenous ENaC with a mutated version of the ENaC gene,or by mutating an endogenous ENaC, e.g., by exposure to known mutagens.

A DNA construct is introduced into the nuclei of embryonic stem cells.Cells containing the newly engineered genetic lesion are injected into ahost mouse embryo, which is re-implanted into a recipient female. Someof these embryos develop into chimeric mice that possess germ cellspartially derived from the mutant cell line. Therefore, by breeding thechimeric mice it is possible to obtain a new line of mice containing theintroduced genetic lesion (see, e.g., Capecchi et al., Science 244:1288(1989)). Chimeric targeted mice can be derived according to Hogan etal., Manipulating the Mouse Embryo: A Laboratory Manual (1988) andTeratocarcinomas and Embryonic Stem Cells: A Practical Approach(Robertson, ed., 1987).

Candidate ENaC Modulators

The compounds tested as modulators of ENaC protein can be any smallorganic molecule, or a biological entity, such as a protein, e.g., anantibody or peptide, a sugar, a nucleic acid, e.g., an antisenseoligonucleotide or a ribozyme, or a lipid. Alternatively, modulators canbe genetically altered versions of an ENaC protein. Typically, testcompounds will be small organic molecules, peptides, lipids, and lipidanalogs. In one embodiment, the compound is a menthol analog, eithernaturally occurring or synthetic.

Essentially any chemical compound can be used as a potential modulatoror ligand in the assays of the invention, although most often compoundscan be dissolved in aqueous or organic (especially DMSO-based) solutionsare used. The assays are designed to screen large chemical libraries byautomating the assay steps and providing compounds from any convenientsource to assays, which are typically run in parallel (e.g., inmicrotiter formats on microtiter plates in robotic assays). It will beappreciated that there are many suppliers of chemical compounds,including Sigma (St. Louis, Mo.), Aldrich (St. Louis, Mo.),Sigma-Aldrich (St. Louis, Mo.), Fluka Chemika-Biochemica Analytika(Buchs Switzerland) and the like.

In one preferred embodiment, high throughput screening methods involveproviding a combinatorial small organic molecule or peptide librarycontaining a large number of potential therapeutic compounds (potentialmodulator or ligand compounds). Such “combinatorial chemical libraries”or “ligand libraries” are then screened in one or more assays, asdescribed herein, to identify those library members (particular chemicalspecies or subclasses) that display a desired characteristic activity.The compounds thus identified can serve as conventional “lead compounds”or can themselves be used as potential or actual therapeutics.

A combinatorial chemical library is a collection of diverse chemicalcompounds generated by either chemical synthesis or biologicalsynthesis, by combining a number of chemical “building blocks” such asreagents. For example, a linear combinatorial chemical library such as apolypeptide library is formed by combining a set of chemical buildingblocks (amino acids) in every possible way for a given compound length(i.e., the number of amino acids in a polypeptide compound). Millions ofchemical compounds can be synthesized through such combinatorial mixingof chemical building blocks.

Preparation and screening of combinatorial chemical libraries is wellknown to those of skill in the art. Such combinatorial chemicallibraries include, but are not limited to, peptide libraries (see, e.g.,U.S. Pat. No. 5,010,175, Furka, Int. J. Pept. Prot. Res. 37:487-493(1991) and Houghton et al., Nature 354:84-88 (1991)). Other chemistriesfor generating chemical diversity libraries can also be used. Suchchemistries include, but are not limited to: peptoids (e.g., PCTPublication No. WO 91/19735), encoded peptides (e.g., PCT PublicationNo. WO 93/20242), random bio-oligomers (e.g., PCT Publication No. WO92/00091), benzodiazepines (e.g., U.S. Pat. No. 5,288,514), diversomerssuch as hydantoins, benzodiazepines and dipeptides (Hobbs et al., Proc.Nat. Acad. Sci. USA 90:6909-6913 (1993)), vinylogous polypeptides(Hagihara et al., J. Amer. Chem. Soc. 114:6568 (1992)), nonpeptidalpeptidomimetics with glucose scaffolding (Hirschmann et al., J. Amer.Chem. Soc. 114:9217-9218 (1992)), analogous organic syntheses of smallcompound libraries (Chen et al., J. Amer. Chem. Soc. 116:2661 (1994)),oligocarbamates (Cho et al., Science 261:1303 (1993)), and/or peptidylphosphonates (Campbell et al., J. Org. Chem. 59:658 (1994)), nucleicacid libraries (see Ausubel, Berger and Sambrook, all supra), peptidenucleic acid libraries (see, e.g., U.S. Pat. No. 5,539,083), antibodylibraries (see, e.g., Vaughn et al., Nature Biotechnology, 14(3):309-314(1996) and PCT/US96/10287), carbohydrate libraries (see, e.g., Liang etal., Science, 274:1520-1522 (1996) and U.S. Pat. No. 5,593,853), smallorganic molecule libraries (see, e.g., benzodiazepines, Baum C&EN,January 18, page 33 (1993); isoprenoids, U.S. Pat. No. 5,569,588;thiazolidinones and metathiazanones, U.S. Pat. No. 5,549,974;pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134; morpholinocompounds, U.S. Pat. No. 5,506,337; benzodiazepines, U.S. Pat. No.5,288,514, and the like).

Devices for the preparation of combinatorial libraries are commerciallyavailable (see, e.g., 357 MPS, 390 MPS, Advanced Chem Tech, LouisvilleKy., Symphony, Rainin, Woburn, Mass., 433A Applied Biosystems, FosterCity, Calif., 9050 Plus, Millipore, Bedford, Mass.). In addition,numerous combinatorial libraries are themselves commercially available(see, e.g., ComGenex, Princeton, N.J., Asinex, Moscow, Ru, Tripos, Inc.,St. Louis, Mo., ChemStar, Ltd, Moscow, RU, 3D Pharmaceuticals, Exton,Pa., Martek Biosciences, Columbia, Md.). C. Solid State and Soluble HighThroughput Assays

Additionally soluble assays can be effected using a ENaC protein, or acell or tissue expressing a ENaC protein, either naturally occurring orrecombinant. Still alternatively, solid phase based in vitro assays in ahigh throughput format can be effected, where the ENaC protein orfragment thereof, such as the cytoplasmic domain, is attached to a solidphase substrate. Any one of the assays described herein can be adaptedfor high throughput screening, e.g., ligand binding, calcium flux,change in membrane potential, etc.

In the high throughput assays of the invention, either soluble or solidstate, it is possible to screen several thousand different modulators orligands in a single day. This methodology can be used for ENaC proteinsin vitro, or for cell-based or membrane-based assays comprising an ENaCprotein. In particular, each well of a microtiter plate can be used torun a separate assay against a selected potential modulator, or, ifconcentration or incubation time effects are to be observed, every 5-10wells can test a single modulator. Thus, a single standard microtiterplate can assay about 100 (e.g., 96) modulators. If 1536 well plates areused, then a single plate can easily assay from about 100-about 1500different compounds. It is possible to assay many plates per day; assayscreens for up to about 6,000, 20,000, 50,000, or more than 100,000different compounds are possible using the integrated systems of theinvention.

For a solid state reaction, the protein of interest or a fragmentthereof, e.g., an extracellular domain, or a cell or membrane comprisingthe protein of interest or a fragment thereof as part of a fusionprotein can be bound to the solid state component, directly orindirectly, via covalent or non covalent linkage e.g., via a tag. Thetag can be any of a variety of components. In general, a molecule whichbinds the tag (a tag binder) is fixed to a solid support, and the taggedmolecule of interest is attached to the solid support by interaction ofthe tag and the tag binder.

A number of tags and tag binders can be used, based upon known molecularinteractions well described in the literature. For example, where a taghas a natural binder, for example, biotin, protein A, or protein G, itcan be used in conjunction with appropriate tag binders (avidin,streptavidin, neutravidin, the Fc region of an immunoglobulin, etc.)Antibodies to molecules with natural binders such as biotin are alsowidely available and appropriate tag binders; see, SIGMA Immunochemicals1998 catalogue SIGMA, St. Louis Mo.).

Similarly, any haptenic or antigenic compound can be used in combinationwith an appropriate antibody to form a tag/tag binder pair. Thousands ofspecific antibodies are commercially available and many additionalantibodies are described in the literature. For example, in one commonconfiguration, the tag is a first antibody and the tag binder is asecond antibody which recognizes the first antibody. In addition toantibody-antigen interactions, receptor-ligand interactions are alsoappropriate as tag and tag-binder pairs. For example, agonists andantagonists of cell membrane receptors (e.g., cell receptor-ligandinteractions such as transferrin, c-kit, viral receptor ligands,cytokine receptors, chemokine receptors, interleukin receptors,immunoglobulin receptors and antibodies, the cadherin family, theintegrin family, the selectin family, and the like; see, e.g., Pigott &Power, The Adhesion Molecule Facts Book I (1993). Similarly, toxins andvenoms, viral epitopes, hormones (e.g., opiates, steroids, etc.),intracellular receptors (e.g. which mediate the effects of various smallligands, including steroids, thyroid hormone, retinoids and vitamin D;peptides), drugs, lectins, sugars, nucleic acids (both linear and cyclicpolymer configurations), oligosaccharides, proteins, phospholipids andantibodies can all interact with various cell receptors.

Synthetic polymers, such as polyurethanes, polyesters, polycarbonates,polyureas, polyamides, polyethyleneimines, polyarylene sulfides,polysiloxanes, polyimides, and polyacetates can also form an appropriatetag or tag binder. Many other tag/tag binder pairs are also useful inassay systems described herein, as would be apparent to one of skillupon review of this disclosure.

Common linkers such as peptides, polyethers, and the like can also serveas tags, and include polypeptide sequences, such as poly gly sequencesof between about 5 and 200 amino acids. Such flexible linkers are knownto persons of skill in the art. For example, poly(ethelyne glycol)linkers are available from Shearwater Polymers, Inc. Huntsville, Ala.These linkers optionally have amide linkages, sulfhydryl linkages, orheterofunctional linkages.

Tag binders are fixed to solid substrates using any of a variety ofmethods currently available. Solid substrates are commonly derivatizedor functionalized by exposing all or a portion of the substrate to achemical reagent which fixes a chemical group to the surface which isreactive with a portion of the tag binder. For example, groups which aresuitable for attachment to a longer chain portion would include amines,hydroxyl, thiol, and carboxyl groups. Aminoalkylsilanes andhydroxyalkylsilanes can be used to functionalize a variety of surfaces,such as glass surfaces. The construction of such solid phase biopolymerarrays is well described in the literature. See, e.g., Merrifield, J.Am. Chem. Soc. 85:2149-2154 (1963) (describing solid phase synthesis of,e.g., peptides); Geysen et al., J. Immunol. Meth. 102:259-274 (1987)(describing synthesis of solid phase components on pins); Frank &Doring, Tetrahedron 44:6031-6040 (1988) (describing synthesis of variouspeptide sequences on cellulose disks); Fodor et al., Science,251:767-777 (1991); Sheldon et al., Clinical Chemistry 39(4):718-719(1993); and Kozal et al., Nature Medicine 2(7):753-759 (1996) (alldescribing arrays of biopolymers fixed to solid substrates).Non-chemical approaches for fixing tag binders to substrates includeother common methods, such as heat, cross-linking by UV radiation, andthe like.

Having described the invention supra, the examples provided infrafurther illustrate some preferred embodiments of the invention. Theseexamples are provided only for purposes of illustration and should notbe construed as limiting the subject invention.

Practical Applications of the Invention

Compounds which modulate, preferably enhance the activity of delta hENaChave important implications in modulation of human salty taste.

Compounds which activate hENaC in taste papillae on the tongue can beused to enhance salt sensation by promoting Na⁺ transport into taste budcells (Kretz et al., J Histochem Cytochem., 4751-64 (1999); Lin et al.,J Comp. Neurol. 405:406-420 (1999). This has obvious consumerapplications in improving the taste and palatability of low salt foodsand beverages.

The following examples were effected using the materials and methodsdescribed supra. These examples are put forth so as to provide those ofordinary skill in the art with a complete disclosure and description ofhow to make and use the subject invention, and are not intended to limitthe scope of what is regarded as the invention.

EXAMPLES Example 1

Comparison of Delta Beta Gamma hENaC and Alpha Beta Gamma mENaC Functionin Oocytes

Oocytes described above expressing delta beta gamma human ENaC and alphabeta gamma human ENaC function in oocytes were produced using thematerials and methods described supra and ENaC sequences provided in theSequence Listing preceding the claims. These oocytes were then contactedwith amiloride in the absence and presence of a proprietary ENaCenhancer identified by Senomyx. As shown in FIG. 1A, representativeamiloride inhibition curves for alpha beta gamma hENaC (n equals 16) anddelta beta gamma hENaC (n equals 10) were obtained. Half-maximalinhibition of delta beta gamma hENaC required about 25-fold higherconcentrations of amiloride compared with alpha beta gamma mENaC. Asshown in FIG. 1B the representative proprietary enhancer compoundidentified as 6363969 resulted in the dose-response curves for alphabeta gamma hENaC (n equals 46) and delta beta gamma hENaC (n equals 11)shown therein. The proprietary compound activated delta beta gamma hENaCwith similar efficacy and potency as alpha beta gamma hENaC. Theexperiments testing compounds with delta beta gamma hENaC used 10micromolar amiloride whereas experiments testing compounds with alphabeta gamma hENaC used 1 micromolar amiloride, concentrations yieldinggreater than 90% hENaC inhibition, to calculate % hENaC activationvalues.

Example 2

Representative Trace Results

The results of other electrophysiological experiments are also shown inFIGS. 2A and 2B. Representative traces of oocytes expressing alpha betagamma hENaC are shown in FIG. 2A (top traces) and FIG. 2B containsresults for delta beta gamma hENaC (bottom traces) and stimulated withamiloride (1 micromolar for alpha ENaC and 10 micromolar for delta ENaC)and further contacted with the proprietary enhancer compound 6363969 (1micromolar concentration). This proprietary compound as shown in FIGS.2A and 2B was found to strongly activate both alpha and delta subunitcontaining hENaC channels. The trace results show current (uA) on they-axis as a function of time (sec) on the x-axis. As shown from theresults in FIGS. 2A and 2B the results for both the alpha and the deltacontaining ENaC are similar.

Example 3

Taste Cell Specific Expression of Delta ENaC Shown by PCR

In order to confirm the potential role of the delta ENaC and other ENaCsubunits in taste perception, particularly salty taste, 73 sodiumchannels were screened from the monkey genome (Macaca fascicularis orcynomolgus macaque) for expression in monkey circumvallate (CV) papillataste cells but not in control lingual epithelial cells by PCR. Bothtaste and lingual cells wee isolated by laser capture microdissection.These PCR experiments identified numerous taste-specific genes asanticipated including the G-protein gustducin, a gene encoding apolypeptide component of the sweet receptor, T1R2, the ion channel TRPM5(expressed in sweet, bitter, and umami cells) and PKD2L1 (an ion channelexpressed in sour cells). Of relevance to the present invention theinventors identified delta ENaC as a taste-specific gene whereas alpha,beta and gamma ENaC were not taste-cell specific but rather wereexpressed in both taste and lingual cells (FIG. 3). As shown thereinmonkey PCR primers specific for each gene were used to amplify cDNA frompurified circumvallate (CV) taste or lingual cells isolated by lasercapture microdissection. In the Figure a ‘+’ indicates that reversetranscription was performed and that cDNA was added to the PCR reaction.A ‘-’ indicates that no reverse transcription was performed and that nocDNA was added to the PCR reaction. As shown in the Figure delta ENaC isonly present in taste cells and is not expressed in lingual cells. DNAsequencing analysis further confirmed the sequences of all four ENaCgenes in taste cells.

Example 4

Taste Cell Specific Expression Shown by In Situ Hybridization

Histology experiments were also performed to determine whether deltaENaC is expressed in a subset of taste receptor cells as would beexpected for a salt taste receptor target. Using in situ hybridizationto label delta ENaC mRNA, it was determined that delta ENaC wasexpressed in a subset of monkey CV cells (See FIG. 4). As shown thereinthe cells identified by the arrows denote taste cells expressing deltaENaC. Only a subset of taste cells express the delta ENaC protein, asexpected for a salt receptor.

Additionally, Table 1 below contains a summary of the results of similarelectrophysiological experiments conducted using the delta beta gammaand alpha beta gamma hENaC which were activated by various enhancerchemical classes. These assays similarly revealed that the alpha betagamma and delta beta gamma ENaC channels are equally stimulated bydifferent enhancer classes. [These experiments used 5-7 oocytes perexperiment.] Based on these results, it is anticipated that delta ENaCenhancers identified using this or similar cell-based assays may be usedto modulate salty taste.

TABLE 1 Summary of αβγ and δβγ hENaC activation by various enhancerchemical classes. Alpha beta gamma and delta beta gamma ENaC channelsare equally stimulated by different enhancer classes. N = 5-7 oocytesper experiment % Enhance % Enhance EC50 (uM) EC50(uM) Compound αβγ δβγαβγ δβγ 6363969 359 +/− 67 348 +/− 94 0.47 0.32 (1 uM) 6028354 376 +/−43 219 +/− 54 1.90 1.02 (3 uM) UGI  29 +/− 12 25 +/− 7 ND ND (100 uM) Choline C1  53 +/− 17 52 +/− 5 ND ND

REFERENCES

All the references cited in this application are incorporated byreference in their entirety herein.

SEQUENCE LISTING OF ENaC DNA AND PROTEIN SEQUENCES SEQUENCE NO: 1:Human Delta ENaC DNA Sequence:atggctgagcaccgaagcatggacgggagaatggaagcagccacacgggggggctctcacctccaggctgcagcccagacgccccccaggccggggccaccatcagcaccaccaccaccacccaaggaggggcaccaggaggggctggtggagctgcccgcctcgttccgggagctgctcaccttcttctgcaccaatgccaccatccacggcgccatccgcctggtctgctcccgcgggaaccgcctcaagacgacgtcctgggggctgctgtccctgggagccctggtcgcgctctgctggcagctggggctcctctttgagcgtcactggcaccgcccggtcctcatggccgtctctgtgcactcggagcgcaagctgctcccgctggtcaccctgtgtgacgggaacccacgtcggccgagtccggtcctccgccatctggagctgctggacgagtttgccagggagaacattgactccctgtacaacgtcaacctcagcaaaggcagagccgccctctccgccactgtcccccgccacgagccccccttccacctggaccgggagatccgtctgcagaggctgagccactcgggcagccgggtcagagtggggttcagactgtgcaacagcacgggcggcgactgcttttaccgaggctacacgtcaggcgtggcggctgtccaggactggtaccacttccactatgtggatatcctggccctgctgcccgcggcatgggaggacagccacgggagccaggacggccacttcgtcctctcctgcagttacgatggcctggactgccaggcccgacagttccggaccttccaccaccccacctacggcagctgctacacggtcgatggcgtctggacagctcagcgccccggcatcacccacggagtcggcctggtcctcagggttgagcagcagcctcacctccctctgctgtccacgctggccggcatcagggtcatggttcacggccgtaaccacacgcccttcctggggcaccacagcttcagcgtccggccagggacggaggccaccatcagcatccgagaggacgaggtgcaccggctcgggagcccctacggccactgcaccgccggcggggaaggcgtggaggtggagctgctacacaacacctcctacaccaggcaggcctgcctggtgtcctgcttccagcaActgatggtggagacctgctcctgtggctactacctccaccctctgccggcgggggctgagtactgcagctctgcccggcaccctgcctggggacactgcttctaccgcctctaccaggacctggagacccaccggctcccctgtacctcccgctgccccaggccctgcagggagtctgcattcaagctctccactgggacctccaggtggccttccgccaagtcagctggatggactctggccacgctaggtgaacaggggctgccgcatcagagccacagacagaggagcagcctggccaaaatcaacatcgtctaccaggagctcaactaccgctcagtggaggaggcgcccgtgtactcggtgccgcagctgctctcGgccatgggcagcctctGcagcctgtggtttggggcctccgtcctctccctcctggagctcctggagctgctgctcgatgcttctgccctcaccctggtgctaggcggccgccggctccgcagggcgtggttctcctggcccagagccagccctgcctcaggggcgtccagcatcaagccagaggccagtcagatgcccccgcctgcaggcggcacgtcagatgacccggagcccagcgggcctcatctcccacgggtgatgcttccaggggttctggcgggagtTtcagccgaagagagctgggctgggccccagccccttgagactctggacacctgaSEQUENCE NO: 2: Human Delta ENaC Protein Sequence:MAEHRSMDGRMEAATRGGSHLQAAAQTPPRPGPPSAPPPPPKEGHQEGLVELPASFRELLTFFCTNATIHGAIRLVCSRGNRLKTTSWGLLSLGALVALCWQLGLLFERHWHRPVLMAVSVHSERKLLPLVTLCDGNPRRPSPVLRHLELLDEFARENIDSLYNVNLSKGRAALSATVPRHEPPFHLDREIRLQRLSHSGSRVRVGFRLCNSTGGDCFYRGYTSGVAAVQDWYHFHYVDILALLPAAWEDSHGSQDGHFVLSCSYDGLDCQARQFRTFHHPTYGSCYTVDGVWTAQRPGITHGVGLVLRVEQQPHLPLLSTLAGIRVMVHGRNHTPFLGHHSFSVRPGTEATISIREDEVHRLGSPYGHCTAGGEGVEVELLHNTSYTRQACLVSCFQQLMVETCSCGYYLHPLPAGAEYCSSARHPAWGHCFYRLYQDLETHRLPCTSRCPRPCRESAFKLSTGTSRWPSAKSAGWTLATLGEQGLPHQSHRQRSSLAKINIVYQELNYRSVEEAPVYSVPQLLSAMGSLcSLWFGASVLSLLELLELLLDASALTLVLGGRRLRRAWFSWPRASPASGASSIKPEASQMPPPAGGTSDDPEPSGPHLPRVMLPGVLAGVSAEESWA GPQPLETLDTSEQUENCE NO: 3: Human Alpha ENaC DNA Sequence:atggaggggaacaagctggaggagcaggactetagccctccacagtccactccagggctcatgaaggggaacaagcgtgaggagcaggggctgggccccgaacctgcggcgccccagcagcccacggcggaggaggaggccctgatcgagttccaccgctcctaccgagagctcttcgagttcttctgcaacaacaccaccatccacggcgccatccgcctggtgtgctcccagcacaaccgcatgaagacggccttctgggcagtgctgtggctctgcacctttggcatgatgtactggcaattcggcctgcttttcggagagtacttcagctaccccgtcagcctcaacatcaacctcaactcggacaagctcgtcttccccgcagtgaccatctgcaccctcaatccctacaggtacccggaaattaaagaggagctggaggagctggaccgcatcacagagcagacgctctttgacctgtacaaatacagctccttcaccactctcgtggccggctcccgcagccgtcgcgacctgcgggggactctgccgcaccccttgcagcgcctgagggtcccgcccccgcctcacggggcccgtcgagcccgtagcgtggcctccagcttgcgggacaacaacccccaggtggactggaaggactggaagatcggcttccagctgtgcaaccagaacaaatcggactgcttctaccagacatactcatcaggggtggatgcggtgagggagtggtaccgcttccactacatcaacatcctgtcgaggctgccagagactctgccatccctggaggaggacacgctgggcaacttcatcttcgcctgccgcttcaaccaggtctcctgcaaccaggcgaattactctcacttccaccacccgatgtatggaaactgctatactttcaatgacaagaacaactccaacctctggatgtcttccatgcctggaatcaacaacggtctgtccctgatgctgcgcgcagagcagaatgacttcattcccctgctgtccacagtgactggggcccgggtaatggtgcacgggcaggatgaacctgcctttatggatgatggtggctttaacttgcggcctggcgtggagacctccatcagcatgaggaaggaaaccctggacagacttgggggcgattatggcgactgcaccaagaatggcagtgatgttcctgttgagaacctttacccttcaaagtacacacagcaggtgtgtattcactcctgcttccaggagagcatgatcaaggagtgtggctgtgcctacatcttctatccgcggccccagaacgtggagtactgtgactacagaaagcacagttcctgggggtactgctactataagctccaggttgacttctcctcagaccacctgggctgfttcaccaagtgccggaagccatgcagcgtgaccagctaccagctctctgctggttactcacgatggccctcggtgacatcccaggaatgggtcttccagatgctatcgcgacagaacaattacaccgtcaacaacaagagaaatggagtggccaaagtcaacatcttcttcaaggagctgaactacaaaaccaattctgagtctccctctgtcacgatggtcaccctcctgtccaacctgggcagccagtggagcctgtggttcggctcctcggtgttgtctgtggtggagatggctgagctcgtctttgacctgctggtcatcatgttcctcatgctgctccgaaggttccgaagccgatactggtctccaggccgagggggcaggggtgctcaggaggtagcctccaccctggcatcctcccctccttcccacttctgcccccaccccatgtctctgtccttgtcccagccaggccctgctccctctccagccttgacagcccctccccctgcctatgccaccctgggcccccgcccatctccagggggctctgcaggggccagttcctccacctgtcctctgggggggccctga SEQUENCE NO: 4: Human Alpha ENaC Protein Sequence:MEGNKLEEQDSSPPQSTPGLMKGNKREEQGLGPEPAAPQQPTAEEEALIEFHRSYRELFEFFCNNTTIHGAIRLVCSQHNRMKTAFWAVLWLCTFGMMYWQFGLLFGEYFSYPVSLNINLNSDKLVFPAVTICTLNPYRYPEIKEELEELDRITEQTLFDLYKYSSFTTLVAGSRSRRDLRGTLPHPLQRLRVPPPPHGARRARSVASSLRDNNPQVDWKDWKIGFQLCNQNKSDCFYQTYSSGVDAVREWYRFHYINILSRLPETLPSLEEDTLGNFIFACRFNQVSCNQANYSHFHHPMYGNCYTFNDKNNSNLWMSSMPGINNGLSLMLRAEQNDFIPLLSTVTGARVMVHGQDEPAFMDDGGFNLRPGVETSISMRKETLDRLGGDYGDCTKNGSDVPVENLYPSKYTQQVCIHSCFQESMIKECGCAYIFYPRPQNVEYCDYRKHSSWGYCYYKLQVDFSSDHLGCFTKCRKPCSVTSYQLSAGYSRWPSVTSQEWVFQMLSRQNNYTVNNKRNGVAKVNIFFKELNYKTNSESPSVTMVTLLSNLGSQWSLWFGSSVLSVVEMAELVFDLLVIMFLMLLRRFRSRYWSPGRGGRGAQEVASTLASSPPSHFCPHPMSLSLSQPGPAPSPALTAPPPAYATLGPRPSPGGSAGASSSTCPLGGP SEQUENCE NO: 5:Human Beta ENaC DNA Sequence:atgcacgtgaagaagtacctGctgaagggcctgcatcggctgcagaagggccccggctacacgtacaaggagctgctggtgtggtactgcgacaacaccaacacccacggccccaagcgcatcatctgtgaggggcccaagaagaaagccatgtggttcctgctcaccctgctcttcgccgccctcgtctgctggcagtggggcatcttcatcaggacctacttgagctgggaggtcagcgtctccctctccgtaggcttcaagaccatggacttccccgccgtcaccatctgcaatgctagccccttcaagtattccaaaatcaagcatttgctgaaggacctggatgagctgatggaagctgtcctggagagaatcctggctcctgagctaagccatgccaatgccaccaggaacctgaacttctccatctggaaccacacacccctggtccttattgatgaacggaacccccaccaccccatggtccttgatctctttggagacaaccacaatggcttaacaagcagctcagcatcagaaaagatctgtaatgcccacgsgtgcaaaatggccatgagactatgtagcctcaacaggacccagtgtaccttccggaacttcaccagtgctacccaggcattgacagagtggtacatcctgcaggccaccaacatctttgcacaggtgccacagcaggagctagtagagatgagctaccccggcgagcagatgatcctggcctgcctattcggagctgagccctgcaactaccggaacttcacgtccatcttctaccctcactatggcaactgttacatcttcaactggggcatgacagagaaggcacttccttcggccaaccctggaactgaattcggcctgaagttgatcctggacataggccaggaagactacgtccccttccttgcgtccacggccggggtcaggctgatgcttcacgagcagaggtcataccccttcatcagagatgagggcatctacGccatgtcggggacagagacgtccatcggggtactcgtggacaagcttcagcgcatgggggagccctacagcccgtgcaccgtgaatggttctgaggtccccgtccaaaacttctacagtgactacaacacgacctactccatccaggcctgtcttcgctcctgcttccaagaccacatgatccgtaactgcaactgtggccactacctgtacccactGccccgtggggagaaatactgcaacaaccgggacttcccagactgggcccattgctactcagatctacagatgagcgtggcgcagagagagacctgcattggcatgtgcaaggagtcctgcaatgacacccagtacaagatgaccatctccatggctgactggccttctgaggcctccgaggactggattttccacgtcttgtctcaggagcgggaccaaagcaccaatatcaccctgagcaggaagggaattgtcaagctcaacatctActtccaagaatttaactatcgcaccattgaagaatcagcagccaataacatcgtctggctgctctcgaatctgggtggccagtttggcttctggatggggggctctgtgctgtgcctcatcgagtttggggagatcatcatcgactttgtgtggatcaccatcatcaagctggtggccttggccaagagcctacggcagcggcgagcccaagccagCtacgctggcccaccgcccaccgtggccgagctggtggaggcccacaccaactttggcttccagcctgacacggccccccgcagccccaacactgggccctaccccagtgagcaggccctgcccatcccaggcaccccgccccccaactatgactccctgcgtctgcagccgctggacgtcatcgagtctgacagtgagggtgatgccatctaa SEQUENCE NO: 6: Human Beta ENaC Protein Sequence:MHVKKYLLKGLHRLQKGPGYTYKELLVWYCDNTNTHGPKRIICEGPKKKAMWFLLTLLFAALVCWQWGIFIRTYLSWEVSVSLSVGFKTMDFPAVTICNASPFKYSKIKHLLKDLDELMEAVLERILAPELSHANATRNLNFSIWNHTPLVLIDERNPHHPMVLDLFGDNHNGLTSSSASEKICNAHGCKMAMRLCSLNRTQCTFRNFTSATQALTEWYILQATNIFAQVPQQELVEMSYPGEQMILACLFGAEPCNYRNFTSIFYPHYGNCYIFNWGMTEKALPSANPGTEFGLKLLILDIGQEDYVPFLASTAGVRLMLHEQRSYPFIRDEGIYAMSGTETSIGVLVDKLQRMGEPYSPCTVNGSEVPVQNFYSDYNTTYSIQACLRSCFQDHMIRNCNCGHYLYPLPRGEKYCNNRDFPDWAHCYSDLQMSVAQRETCIGMCKESCNDTQYKMTISMADWPSEASEDWIFHVLSQERDQSTNITLSRKGIVKLNIYFQEFNYRTIEESAANNIVWLLSNLGGQFGFWMGGSVLCLIEFGEIIIDFVWITIIKLVALAKSLRQRRAQASYAGPPPTVAELVEAHTNFGFQPDTAPRSPNTGPYPSEQALPIPGTPPPNYDSLRLQPLDVI ESDSEGDAISEQUENCE NO: 7: Human Gamma ENaC DNA Sequence:atggcacccggagagaagatcaaagccaaaatcaagaagaatctgcccgtgacgggccctcaggcgccgaccattaaagagctgatgcggtggtactgcctcaacaccaacacccatggctgtcgccgcatcgtggtgtcccgcggccgtctgcgccgcctcctctggatcgggttcacactgactgccgtggccctcatcctctggcagtgcgccctcctcgtcttctccttctatactgtctcagtttccatcaaagtccacttccggaagctggattttcctgcagtcaccatctgcaacatcaacccctacaagtacagcaccgttcgccaccttctagctgacttggaacaggagaccagagaggccctgaagtccctgtatggctttccagagtcccggaagcgccgagaggcggagtcctggaactccgtctcagagggaaagcagcctagattctcccaccggattccgctgctgatctttgatcaggatgagaagggcaaggccagggacttcttcacagggAggaagcggaaagtcggcggtagcatcattcacaaggcttcaaatgtcatgcacatcgagtccaagcaagtggtgggattccaactgtgctcaaatgacacctccgactgtgccacctacaccttcagctcgggaatcaatgccattcaggagtggtataagctacactacatgaacatcatggcacaggtgcctctggagaagaaaatcaacatgagctattctgctgaggagctgctggtgacctgcttctttgatggagtgtcctgtgatgccaggaatttcacgcttttCcaccacccgatgcatgggaattgctatactttcaacaacagagaaaatgagaccattctcagcacctccatggggggcagcgaatatgggctgcaagtcattttgtacataaacgaagaggaatacaacccattcctcgtgtcctccactggagctaaggtgatcatccatcggcaggatgagtatcccttcgtcgaagatgtgggaacagagattgagacagcaatggtcacctctataggaatgcacctgacagagtccttcaagctgagtgagccctacagtcagtgcacggaggacgggagtgacgtgccaatcaggaacatctacaacgctgectactcgctccagatctgccttcattcatgcttccagacaaagatggtggagaaatgtgggtgtgcccagtacagccagcctctacctcctgcagccaactactgcaactaccagcagcaccccaactggatgtattgttactaccaactgcatcgagcctttgtccaggaagagctgggctgccagtctgtgtgcaaggaagcctgcagctttaaagagtggacactaaccacaagcctggcacaatggccatctgtggtttcggagaagtggttgctgcctgttctcacttgggaccaaggccggcaagtaaacaaaaagctcaacaagacagacttgGccaaactcttgatattctacaaagacctgaaccagagatccatcatggagagcccagccaacagtattgagatgcttctgtccaacttcggtggccagctgggcctgtggatgagctgctctgttgtctgcgtcatcgagatcatcgaggtcttcttcattgacttcttctctatcattgcccgccgccagtggcagaaagccaaggagtggtgggcctggaaacaggctcccccatgtccagaagctccccgtagcccacagggccaggacaatccagccctggatatagacgatgacctacccactttcaactctgctttgcacctgcctccaGccctaggaacccaagtgcccggcacaccgccccccaaatacaataccttgcgcttggagagggccttttccaaccagctcacagatacccagatgctAgatgagctctga SEQUENCE NO: 8:Human Gamma ENaC Protein Sequence:MAPGEKIKAKIKKNLPVTGPQAPTIKELMRWYCLNTNTHGCRRIVVSRGRLRRLLWIGFTLTAVALILWQCALLVFSFYTVSVSIKVHFRKLDFPAVTICNINPYKYSTVRHLLADLEQETREALKSLYGFPESRKRREAESWNSVSEGKQPRFSHRIPLLIFDQDEKGKARDFFTGRKRKVGGSIIHKASNVMHIESKQVVGFQLCSNDTSDCATYTFSSGINAIQEWYKLHYMNIMAQVPLEKKINMSYSAEELINTCFFDGVSCDARNFTLFHHPMHGNCYTFNNRENETILSTSMGGSEYGLQVILYINEEEYNPFLVSSTGAKVIIHRQDEYPFVEDVGTEIETAMVTSIGMHLTESFKLSEPYSQCTEDGSDVPIRNIYNAAYSLQICLHSCFQTKMVEKCGCAQYSQPLPPAANYCNYQQHPNWMYCYYQLHRAFVQEELGCQSVCKEACSFKEWTLTTSLAQWPSVVSEKWLLPVLTWDQGRQVNKKLNKTDLAKLLIFYKDLNQRSIMESPANSIEMLLSNFGGQLGLWMSCSVVCVIEIIEVFFIDFFSIIARRQWQKAKEWWAWKQAPPCPEAPRSPQGQDNPALDIDDDLPTFNSALHLPPALGTQVPGTPPPKYNTLRLERAFSNQLTDTQMLDEL

1-29. (canceled)
 30. A method for identifying compounds having potentialin vivo application for modulating taste comprising: (i) contacting acell that expresses a nucleic acid encoding a human epithelial sodiumchannel (“hENaC”) channel comprising a delta subunit polypeptidepossessing at least 90% sequence identity to the polypeptide identicalto the polypeptide of SEQ ID NO: 10 with one or more putative hENaCenhancer compounds; (ii) conducting an electrophysiological assay whichdetects sodium conductance in the presence and absence of said one ormore putative hENaC enhancer compounds; (iii) based on the results ofstep (ii) identifying the compound as putatively modulating human tasteif the compound increases sodium conductance; and (iv) assaying theeffect(s) of one or more compounds or derivatives thereof which areidentified in step (iii) to increase sodium conductance in a taste test.31. The method of claim 30, wherein the taste test is effected in ahuman subject.
 32. The method of claim 32, wherein the taste testdetermines the effect if any of the compound or a derivative thereof onsalty taste perception.
 33. The method of claim 30, wherein the cellfurther expresses human beta and gamma hENaC subunits or variantsthereof.
 34. The method of claim 30, wherein the delta hENaC isexpressed in an Xenopus oocyte or a mammalian cell.
 35. The method ofclaim 34, wherein said mammalian cell is selected from the groupconsisting of a Swiss3T3, CHO, BHK, NIH3T3, and a COS cell.
 36. Themethod of claim 30, wherein said electrophysiological assay of (ii)comprises the use of a sodium or voltage sensitive dye.
 37. The methodof claim 30, wherein said electrophysiological assay of (ii) is a patchclamp or two electrode voltage clamp assay.
 38. The method of claim 36,wherein said sodium or voltage sensitive dye is selected from the groupconsisting of voltage-sensitive blue dye,4-(2-(6-(dibutylamino)-2-naphthalen-yl)ethenyl)-1-(3-sulfopropyl)hydroxide,inner salt, DiSBACC4(2)(bis-(1,2-dibabituric acid)-triethine oxanol),Cc-2-DMPE, 1,2-ditetradecanoyl-sn-glycero-3-phosphoethanolamine,triethylammonium salt and SBFI-AM (1,3-benzenedicarboxylic acid,4,4-(1,4,10-trioxa-7,13-diazacylopentadecane-7,13-diyibis(5-methoxy-6,1,2-berizofurandiyl))bis-tetrakis[(acetyloxy)methyl]ester.39. The method of claim 30, wherein said electrophysiological assay of(ii) detects delta hENaC activity by an ion flux assay.
 40. The methodof claim 39, wherein said ion flux is detected by atomic absorptionspectroscopy.
 41. The method of claim 30, wherein the delta hENaCsubunit is expressed under the control of a regulatable promoter. 42.The method of claim 30, wherein detection of hENaC activity in step (ii)comprises the use of a fluorescence plate reader or by the use of avoltage imaging plate reader.
 43. The method of claim 30, wherein theeffect of the selected putative enhancer compound on sodium iontransport into taste bud cells is further assayed.
 44. The method ofclaim 42, wherein detection of hENaC activity comprises the use of amembrane potential dye selected from the group consisting ofvoltage-sensitive blue dye, Di-4-ANEPPS (pyridinium,4-(2-(6-(dibutylamino)-2-naphthalen-yl)ethenyl)-1-(3-sulfopropyl)-hydroxide,inner salt); DiSBACC4(2)(bis-(1,2-dibarbituric acid)-trimethine oxanol);DiSBAC4(3) (bis-(1,3-dibarbituric acid)-trimethine oxanol); CC-2-DPME(1,2-dietradecanoyl-sn-glycerol-3-phosphoethanolamine, triethylammoniumsalt) and SBFI-AM (1,3-Benzenedicarboxylic acid,4,4′-[1,4,10-trioxa-7,13-diazacyclopentadecane-7,13-diylbis(5-methoxy-6,1,2-benzofurandiyl)]bis-tetrakis[(acetyloxy)methyl]ester.45. The method of claim 30, wherein said cell stably or transientlyexpresses said delta hENaC.
 46. The method of claim 30, wherein in theelectrophysiological assay in step (ii) delta hENaC activity is effectedusing a Xenopus oocyte that expresses said delta hENaC by patch clampingor two electrode voltage clamping.
 47. The method of claim 30, whereinsaid delta subunit polypeptide possesses at least 95% sequence identityto the polypeptide identical to SEQ ID NO:
 10. 48. The method of claim30, wherein said nucleic acid encoding said delta subunit polypeptidepossesses a single nucleotide polymorphism (“SNP”).